Structure/Function Analysis of Phagocyte Proteins

描述(申请人提供)：吞噬细胞呼吸爆发氧化酶 产生超氧阴离子自由基在宿主防御中起核心作用 炎症反应。活性氧化酶复合体的组装需要 膜蛋白和胞质蛋白的参与，并受 小的GTP酶。一种吞噬细胞特异的b型黄染色质异二聚体，位于 在血浆和中性粒细胞中，特定的颗粒膜是焦点 用于氧化酶组装的点，包含黄素和血红素氧化还原中心 用于将电子从NADPH转移到分子氧。遗传性缺陷 包括两个黄色细胞色素亚基在内的氧化酶蛋白会导致慢性 肉芽肿病(CGD)，一种以无呼吸道为特征的综合征 破裂、反复感染和慢性肉芽肿。结构和 各种氧化物酶亚基和组装之间的功能关系 对活性NADPH氧化酶复合体的研究尚不完全清楚。这 低电位黄色素是一种由gp91Phox组成的杂二聚体，它是一个91 kDa的 由X连锁基因编码的糖蛋白，该基因是 X-连锁的CGD和p221-Phox，这是一种来自于 常染色体CGD基因座。拟议的研究采用了遗传方法来研究 黄细胞色素b和黄色素氧化酶的结构和功能研究 它的作用是组装具有活性的NADPH氧化酶复合体。这个 项目有3个具体目标，这3个目标利用了gp91Phox 基因打靶和基因打靶建立的缺陷吞噬细胞系 我们为表达功能而开发的异源细胞系统 重组氧化酶亚基。首先，gp91Phox和gp91Phox的相对作用 P22Phox在黄细胞素b的生物合成和功能中的作用将用 异源细胞，用于表达未组装的亚基，否则 吞噬细胞不稳定。第二，gp91Phox和p22中结构域的鉴定 在组装和调节氧化酶活性中起作用的PHOX将 采用了一种强调定点突变的策略 候选亲水区的表达和功能分析 在完好无损的细胞中。第三，黄细胞色素b的重要功能结构域 将调查吞噬过程中的运输和NADPH氧化酶组装 在异源和吞噬细胞系中使用类似的方法。这些 研究将提供对超氧化物生成系统的进一步洞察 吞噬细胞，这可能导致调节超氧化物的新方法 在炎症反应和宿主防御中形成。

DESCRIPTION (provided by applicant): The phagocyte respiratory burst oxidase that generates the superoxide radical plays a central role in host defense and the inflammatory response. Assembly of the active oxidase complex requires the participation of both membrane and cytosolic proteins, and is regulated by small GTPases. A phagocyte-specific b-type flavocytochrome heterodimer, located in the plasma and, in neutrophils, specific granule membranes, is the focal point for oxidase assembly, and contains both the flavin and heme redox centers for transfer of electrons from NADPH to molecular oxygen. Genetic defects in oxidase proteins, including the two flavocytochrome subunits, result in chronic granulomatous disease (CGD), a syndrome characterized by an absent respiratory burst, recurrent infections, and chronic granulomas. The structural and functional relationships between the various oxidase subunits and the assembly of the active NADPH oxidase complex remain incompletely understood. This low-potential flavocytochrome is a heterodimer comprised of gp91 phox, a 91-kDa glycoprotein encoded by an X-linked gene that is the site of mutations in X-linked CGD, and p221-phOx, a non-glycosylated peptide derived from an autosomal CGD locus. The proposed studies take a genetic approach to investigating the structure and function of the oxidase flavocytochrome b and its role as a focal point for assembly of the active NADPH oxidase complex. The project has 3 specific objectives, which take advantage of a gp91 phox deficient phagocyte cell line developed by gene targeting as well as heterologous cell systems we have developed for expression of functional recombinant oxidase subunits. First, the relative roles of gp91 phox and p22phox in flavocytochrome b biosynthesis and function will be examined using heterologous cells for expression of unassembled subunits, which are otherwise unstable in phagocytes. Second, identification of domains in gp91 phox and p22 phox that function in the assembly and regulation of oxidase activity will pursued, using a strategy that emphasizes site-directed mutagenesis of candidate hydrophilic regions followed by expression and analysis of function in intact cells. Third, functional domains important for flavocytochrome b trafficking and NADPH oxidase assembly during phagocytosis will be investigated using similar approaches in heterologous and phagocytic cell lines. These studies will provide further insight into the superoxide-generating system of the phagocyte, which may lead to new approaches in modulating superoxide formation in the inflammatory response and host defense.